It is becoming increasingly clear that guanine nucleotide binding regulatory proteins are involved in mediating a wide variety of signal transduction processes. However, a detailed mechanistic understanding of these systems has been plagued by a number of factors including the difficulties in isolating the individual components of such systems and the lack of proper physical-biochemical approaches to study these systems under in vivo-like conditions (i.e. in a lipid milieu). The retinal visual transduction system now provides an attractive model for studying signal transfer since all of its primary components can be completely purified in mg quantities. This proposal will describe studies aimed at exploiting the advantages of this retinal system to probe the molecular mechanisms by which nucleotide binding regulatory proteins mediate signal transfer, using fluorescence spectroscopy and reconstitution approaches. These studies are divided into three specific aims: 1) The characterization of the activation of the retinal nucleotide regulatory protein, transducin, by guanine nucleotides, Mg2+, and the photoreceptor, rhodopsin, using fluorescence spectroscopy, 2) The characterization of transducin activation, stimulation of effector (phosphodiesterase) activity, and deactivation (GTPase activity) through steady state kinetic studies in phospholipid vesicle systems, and 3) Structure-function studies aimed at characterizing transducin-effector interactions and, at probing the relative location of the effector coupling site and other important regions on the transducin molecule, using chemical modification and fluorescence techniques. Amoung the specific questions which will be addressed in these studies are: a) how guanine nucleotides and Mg2+ activate nucleotide regulatory proteins in a lipid milieu (i.e. via conformational changes and/or subunit dissociation of the heterotrimeric regulatory proteins), b) what are the specific roles of receptor proteins in the activation process, and c) what is the molecular basis of nucleotide regulatory protein-effector interactions? The results of these studies should be relevant not only to the molecular aspects of vertebrate vision but also to a variety of other types of cellular effector systems that are known, or likely, to be regulated by nucleotide binding regulatory proteins such as adenylate cyclase activity, phosphatidylinositol turnover, Ca2+ mobilization, and growth factor action.